Project Summary/Abstract A strong team of tumor immunologists and Positron Emission Tomography (PET) tracer imaging scientists will test the hypothesis that treatment induced elevation of tumor-associated dendritic cell (DC) over macrophages (M?) (DC/M? ratios) as quantified by immunoPET is a predictor of T cell activation and tumor rejection. Current immunotherapies are effective in some patients with existing anti-tumor immunity, but most are not responsive, showing the pressing need for immune priming and monitoring strategies. CD103+DC are the only known antigen presenting cells (APC) capable of cross-presenting tumor antigens to CD8 T cells, but tumor infiltrates contain mostly immuno-suppressive M?. Our goal is to enable in situ immune priming by overriding intratumoral M? with activated DC and quantify these changes with immunoPET. In BALB NeuT mice that develop spontaneous mammary and salivary tumors, we observed by immunoPET a 2 fold increase in 89Zr ?-CD3 mAb retention in a tumor undergoing immune-mediated partial regression, indicating elevated T cell infiltration. T cell images verified treatment outcome and the need for a salvage regimen, such as check-point blockade. But T cell infiltration is the end stage of a long immune- priming process when the tumor fate is sealed. Treatment outcome may be predicted and treatment plan adjusted, however, at the start of immunotherapy by the relative abundance of DC: M? measured with immunoPET. We show complete or partial tumor regression by intratumoral electroporation of cyclic di-nucleotides (CDN), 3'3'-cGAMP (cyclic [G(3',5')pA(3',5')p], in HER2/neu vaccinated mice. Following this finding, we will combine pFlt-3L and CDN electroporation in the tumor to expand and activate DC. To ensure STING activation, inhibitors of STING negative regulators will be tested in combination. Tumor-associated DC and M? will be quantified before and after treatment, using 18F-?-F4/80 Fab (~50kD, t1/2 ~110 minutes) that binds M?, followed by 64Cu-?-CD11c F(Ab')2 (110kD, t1/2 ~12.7 h) that binds both DC and M? (DC+M?). 18F and 64Cu retention will be expressed as % injected dose/gram tissue (% ID/g). An increase in ?-CD11c, but not ?-F4/80 tracer retention would indicate selective elevation of tumor DC. Specifically, we will Aim 1: Measure DC expansion and activation after intratumoral electroporation of pFlt-3L DNA and cyclic di- nucleotides (CDN), with concomitant inhibition of STING negative regulators, to correlate with tumor immunity. Aim 2: Quantify treatment-induced changes in tumor-associated DC versus macrophages by immunoPET imaging to correlate with tumor immunity. Positive findings would support developing DC:M? immunoPET to guide patient treatment plan.